We have discovered a single estrogen receptor (ERa) mutant in 30% of breast hyperplasias and a majority of invasive breast cancers. This mutation causes an amino acid change in the ER hinge domain (lysine 303 to arginine, K303R), makes the receptor hypersensitive to very low estrogen, increases its transcriptional activity, and increases its stability. The mutation arises somatically in the breast; it has so far never been found in normal tissues. We hypothesize that by affecting key regulatory mechanisms, this specific ER mutation contributes to the development of many premalignant breast lesions, facilitates their progression to cancer, and increases breast cancer cell proliferation and worsens clinical prognosis. To test these hypotheses and explore their clinical significance, we propose: (1) To determine whether the presence of the K303R ER mutant is prognostic for recurrence of primary breast cancer, using our clinical breast tumor bank with extended clinical followup. (2) To determine how the K303R mutant is associated with the progression of hyperplasia and DCIS toward invasive breast cancer, using our unique collection of paired concurrent pre-invasive lesions and invasive breast cancer from the same breasts. (3) To further define the molecular mechanisms underlying the hypersensitive ER mutant phenotype, examining: a) the association of mutant vs. wild-type ER with critical coactivators and co-repressors, using an optical Biosensor, yeast two hybrid screens, and transcriptional transactivation assays; b) the dynamics of mutant vs. wild-type ER/co-regulator complexes on specific promoter DNAs, using ChIP and co-immunoprecipitation assays; and c) altered ubiquitin-mediated degradation of the mutant ER/co-activator complex and its association with loss of the wild-type K303 acetylation site. (4) To directly demonstrate in vivo in xenografl models the biological consequences of the K303R ER mutation by assessing its ability in stable transfectants to: a) enhance the growth potential of ER-positive breast cancer cells; and b) enhance the tumorigenic potential of MCF-10AT breast epithelial cells, which normally progress slowly by defined stages to invasive cancer. These experiments will begin to evaluate the potential clinical significance of the K303R ER mutation as an indicator of poor prognosis in primary breast cancer and of more likely progression to invasive cancer in hyperplasia and DCIS, as well as directly demonstrating the biological effects of the mutant in both malignant and premalignant breast cells. The proposed experiments will also evaluate the mechanisms underlying the mutant's phenotype for possible therapeutic targets, since K303R is so frequent especially in the most ominous breast cancers and perhaps in the most ominous premalignant breast lesions as well.